JAK2 p.(V617F) mutation in Tunisian myeloproliferative neoplasms and its genotype-phenotype correlation

Myeloproliferative neoplasms (MPNs) comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The relationship between JAK2 p.(V617F) mutation and MPNs was first described in 2005. The purpose of this study was to determine the prevalence of JAK2 p.(V617F) mutation in Tunisian patients assessed for MPNs and try to set a genotype-phenotype correlation. A retrospective study was conducted between January 2015 and April 2019. We collected the clinical data of all patients with MPNs suspicion or atypical splanchnic vein thrombosis (SVT). JAK2 p.(V617F) mutation was detected by allele specific real-time quantitative fluorescence PCR (AS-qPCR). We gathered 974 patients who underwent molecular analysis, 55.5% of them were male and 44.5% were female. The median age of all studied patients was 56 years. JAK2 p.(V617F) was found in 349 (35.8%) of total enrolled cases. It was reported in 44%, 37%, 29% and 25% of all patients diagnosed as having respectively ET, PV, PMF and atypical SVT. JAK2 p.(V617F) was negative in 62.2% of patients addressed for suspicion of PV. There was a significant positive correlation between the JAK2 p.(V617F) mutation status, age, gender, white blood cell counts and platelet counts. To our best knowledge, this is the first vast investigation of JAK2 p.(V617F) variant in Tunisia and North Africa with the lowest mutation rate in entire cohort and MPNs subgroups, underlying a specific presentation of this mutation. It is considered as an essential marker of MPNs’ diagnosis and prognosis and is associated with differences in the phenotype of these disorders, helpful for the follow-up of these patients.


Introduction
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of disorders which arise from genetically altered myeloid stem or progenitor cells. MPNs are characterized by an over production of mature myeloid cells. Classical MPNs have been classified into 3 entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) [1]. It has long been recognized that these diseases are characterized by shared clinical, pathological and molecular features. All MPNs entities arise from a single somatically mutated stem cell that clonally expands and results in a single or multilineage hyperplasia.
The year 2005 was a watershed in understanding of the molecular pathogenesis of MPNs with the identification of an acquired recurrent somatic mutation in the Jak homology domain 2(JH2) of Janus kinase 2 (JAK2) gene in a significant proportion of patients with MPNs [2]. This variant represents single nucleotide substitution of G to T at nucleotide 1849 (c.1849G>T) in exon 14 of JAK2 gene, resulting in the substitution of Valine to Phenylalanine at codon 617 p.(V617F) [3]. This dominant gain of function mutation affects auto inhibitory JH2 pseudokinase domain of the JAK2 protein, leading to constitutive activation of JAK2 and JAK/STAT signaling pathway in the absence of ligands [4]. JAK2p.(V617F) is detected in 50-60% of ET and PMF patients and up to 97% of PV patients [5]. Infrequent occurrence of this mutation has been reported in acute leukemia, myelodysplasic syndrome (MDS) and atypical chronic myeloid leukemia [6]. In 2008, the world health organization (WHO) classification recognized the JAK2p.(V617F) mutation as a major criterion for the diagnosis of MPNs [7]. To our best knowledge, one Tunisian publication studied JAK2p.(V617F) mutations in a cohort of 45 patients [8]. The aim of this study is to report the prevalence of JAK2p.(V617F) mutation in MPNs Tunisian patients and try to set a genotypephenotype correlation.

Methods
Ethics statement: this study was approved by the regional committee for medical and health research ethics of Pasteur Institute of Tunis. Written informed consents were obtained from each patient for genetic analysis and publication. No commercial support was involved in this study.
All data analyzed in this study are included in this article.
Study population: a retrospective study was conducted in the laboratory of hematology of Pasteur Institute of Tunis between January 2015 and April 2019. We included all the patients with clinical suspicion of MPN or atypical splanchnic vein thrombosis for whom JAK2 p.(V617F) was prescribed by a clinical hematologist. We gathered clinical information in the prescription of all screened patients: age, gender and the applied classification and diagnostic criteria proposed by the World Health Organization (WHO) in 2008 and the updated version 2016 relative to MPNs [9]. We excluded patients with incomplete clinical data. We performed an Allele specific real-time quantitative fluorescence PCR (AS-qPCR) using ipsogen JAK2 MutaSearch Kit, designed for the specific and quantitative determination of DNA copy number of JAK2p.(V617F) mutation and JAK2 wild type with respective primers [10]. p.(V617F) positive control (PC-V617F JAK2), negative control (NC-V617F JAK2), cut-off sample (COS-V617F JAK2) were provided by the kit. A 96-well plate qPCR device were used and all measures were analysed in duplicate. For one reaction, the mix contained 12.5 μL of TaqMan Universal PCR Master Mix, 1μL of specific primers and probe and 6.5 μL of H2O. Five μL of DNA samples were added to 20 μL of the corresponding qPCR premix well (V617F or WT). Five μL of each control and COS-V617F JAK2 were added in each corresponding 20 μL qPCR premix well. The following cycling conditions were run in the 7500 Sequence Detection System (ABI, Melbourne, Australia) to amplify in real time the increase in fluorescence generated: One cycle at 50°C for 2 minutes of incubation, followed by a 10minute incubation at 95°C, then 50 cycles at 95°C (15 seconds), and an extension step at 63°C (1 minute). The point at which the fluorescent signal rises above the background called crossing threshold (CT), is directly proportional to the quantity of starting DNA. The average CT value obtained (CT V617F and CT WT) for each sample (controls, cut-off sample and patients samples) were calculated. The load limit verified that the patient DNA sample used for the test has been correctly manipulated to ensure the reliability of the final results for JAK2 V617F status. Then we calculated the ΔCT value for all valid samples (ΔCT Sample) and controls (ΔCT PC-V617F, ΔCT NC-V617F, and ΔCT COS) in order to obtain the ΔΔCT value for each patient sample (ΔΔCT Sample) and each control (ΔΔCT PC-V617F and ΔΔCT NC-V617F). The JAK2 V617F mutation was detected if ΔΔCT Sample > + ΔCT COS x 0.07 and was absent if ΔΔCT Sample < -ΔCT COS x 0.07. Statistical analysis: descriptive and comparative statistical analysis were performed using the statistical package SPSS for windows, version 16.
The tests used for comparison of qualitative variables were Chi-squared test (χ2 test). The tests used for comparison of distributions of quantitative variables were Student's T test and Mann-Whitney U test. The degree of significance "p" retained for all the tests was 0.05.
Ethics in publishing: this study was approved by the regional committee for medical and health research ethics of Pasteur Institute of Tunis.

Results
Population basic characteristics: we tested 974 Tunisian patients for JAK2p.(V617F) mutation with clinical suspicion of MPN or a typical splanchnic vein thrombosis. The median age of all studied patients was 56 years with extremes ranging from 1 to 90 years and M/F sex-ratio was 541/433. From the total studied population, the MPNs were divided into: 29.4% (286/974) PV, 28.2% (275/974) ET and 9.2% (89/974) PMF. Unclassified MPNs were found in 25.5 % (249/974) of all cases and 7,7% (75/974) patients were addressed for SVT. We did not report any secondary polycythemia. The clinical and biological characteristics in all tested Tunisian patients are summarized in Table 1. We compared the subgroup with mutant variant to wild type subgroup and found a statistically significant difference in the distribution of age and sex ratio (p= 0.000) ( Table 1). Mean age was significantly higher with JAK2p.(V617F) mutation in total cohort, three subgroups of MPNs and unclassified MPNs (p=0.000-0.032) ( Table 1, Table 2) except in SVT cohort (p=0.856). Age distribution depending on the status of JAK2 in the different subgroups is represented on Figure 1.
In PV cohort, we noticed that patients with JAK2p.(V617F) had significantly higher mean age (p=0.000) and higher platelet counts (p=0.000) than in wild type group. Men were more concerned with JAK2 wild type (p=0.000) ( Table 2). In ET, PMF and SVT subgroups, the mutational status showed no significant correlation with gender, mean levels of hemoglobin, WBC and platelet counts ( Table 2). The mutant status in unclassified MPNs presented a higher level of hemoglobin, statistically significant (p=0.007).

Discussion
In the present study, we determined the prevalence of JAK2 p.(V617F) mutation in the first large Tunisian patients cohort examined for suspected MPNs or SVT. We also investigated the relationship between this mutation and its influence in hemogram variation in 974 patients divided into different subgroups of MPNs.
Myeloproliferative neoplasms mainly occur in adults and only infrequently in children [11]. Patients with MPNs experience a chronic disease with an insidious onset that often lasts many years, during which they may variably progress to acute leukemia or bone marrow failure [12]. The identification of JAK2 p.(V617F) mutation is central to the diagnosis of a clonal process and exclusion of non-neoplastic disorders [13]. In 2008, WHO classification mentioned the positivity of the recurrent somatic mutation in exon 14 of JAK2 gene, as a major criterion of MPNs´ diagnosis specifically ET, PV and PMF. It is detected in around 50-60% of ET and PMF patients and in more than 95% of patients with PV [14].
Polycythemia is a collective designation for disorders with elevated haematocrit and a distinction is made between absolute and relative polycythemias [32]. Secondary polycythemia are absolute polycythemias that are often due to hypoxia related to underlying heart and lung disease, which was not reported in this study [33]. ET is characterized by an increased platelet count with a megakaryocytic hyperplasia, whereas PMF is a more heterogeneous disorder both by its clinical and biological characteristics, defined by the presence of bone marrow fibrosis and megakaryocytic hyperplasia [34,35]. In many cases, a continuous between these disease subtypes can be observed, as documented by the progression of ET and PV to secondary myelofibrosis. Boundaries between these disorders cannot be well established and precise diagnosis at disease onset is often challenging. The use of allele specific realtime quantitative fluorescence PCR has significantly increased our ability to detect small amount of mutated clones [36].
Despite advances in understanding MPNs biology, an important fundamental question of how the same genetic event can be associated with distinct clinical phenotypes remains unresolved, as it is for JAK2 p.(V617F) mutation. The mutational landscape in MPN is much more complex than initially thought [37]. A combination of multiple genetic mutations and a complex interplay of inherited genetic modifiers are likely to be of substantial importance, followed by gender, age and microenvironment disruption [29]. In our large cohort, the allele specific real-time quantitative fluorescence PCR in JAK2 p.(V617F) was informative. However, our study suffers from limitations and bias since we only search for JAK2 p.(V617F). We should consider these data in MPN with caution since JAK2 mutation in exon 12, MPL and CALR mutations were not investigated and further additional studies should test them in the large proportion of the JAK2 negative patients.

Conclusion
From our first large study on Tunisian patients with MPNs and SVT, we have settled the prevalence of JAK2p.(V617F) mutation in our population and found the lowest mutation rate of this variant in all broad cohorts. Unbalanced rates in different subgroups of MPNs were highlighted. The allelespecific quantitative real-time fluorescence PCR in JAK2p.(V617F) keeps its place in routine. The clinical and biological characteristics established in this study could be helpful for the clinicians during the diagnosis and the follow-up of MPNs patients. Furthermore, we must also refine the mutational spectrum of MPNs in Tunisia with additional studies of others driver mutations.

What is known about this topic
 Molecular genetics is a cornerstone in the diagnosis and follow-up of MPN subgroups.

Acknowledgments
We are grateful to all Tunisian clinician hematologists who referred their patients to our department and thankful for all patients´ participation. Special thanks to editors and reviewers for their recommendations and corrections. Table 1: basic clinical and hematological characteristics in negative and positive JAK2p.(V617F) mutation in all tested Tunisian patients Table 2: genotype-phenotype correlation in the different subgroups of myeloproliferative neoplasms in Tunisia  Table 3: associations of JAK2 p.(V617F) mutation with hemogram variations in different subgroups of myeloproliferative neoplasms in some reported studies and Tunisian patients